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The brains of male Fisher 344 rats bearing 80-150 mg intracerebral 9L/Ro tumors were irradiated with doses of 1,250-5,000 rads of x- or gamma-rays. At various times after irradiation, the cerebellum and tumor were excised, dissociated into single cells and the DNA from these cells sedimented through alkaline sucrose gradients in zonal rotors with slow gradient reorienting capability. Quantitation of the DNA repair kinetics demonstrated that the process in both tumor cells and neurons has a fast and slow phase. Although all other alternatives cannot be completely eliminated, we suggest that these two phases are most reasonably interpreted as representing repair of lesions in very accessible and less accessible regions of the genome rather than 1) repair of different types of lesions such as single- or double-strand breaks or 2) removal of immediate breaks and breaks induced during excision repair of latent base damage. The slow repair phase is saturable, but not inducible in both tumor cells and neurons. The data suggest that tumor cells restore their chromosomal DNA structure to the unirradiated state faster than neurons because 1) they contain more of the repair system per unit of DNA and 2) a larger proportion of their genetic material is comprised of very accessible regions. The data also suggest that the entire tumor cell genome may be accessible to the repair enzyme(s), while it is possible that a portion of the neuronal genome may be completely inaccessible.

Biochemical kinetic analyses of the ATP stimulation of the A2 form of calf DNA alpha-polymerase show that when DNA or primer termini are the variable substrates, maximum reaction velocity is independent of ATP concentration. When dNTP concentration is the variable substrate, the apparent Km is invariant with ATP. Such results indicate that the increase in the synthetic rate caused by ATP results from an improvement in synthesis initiation at primer termini. The effect of ATP on the DNA binding affinity of alpha-A2-polymerase was examined by using column chromatography. Passage of the polymerase through native DNA-cellulose at 70 mM ionic strength resulted in 40% binding of the enzyme. In the presence of 4 mM ATP, binding increased to 80%. In both cases, the bound polymerase could be eluted by a 370 mM ionic strength wash. An elution profile similar to that observed in the absence of ATP was obtained with 0.1 mM ATP, 4 mM GTP, or 4 mM each of the nonhydrolyzable ATP analogues adenyl-5'-yl imidodiphosphate or adenosine 5'-O-(3-thiotriphosphate). These results suggest that hydrolysis of the gamma-phosphate occurs at millimolar levels of ATP and leads to a higher affinity of polymerase for DNA. To distinguish the effects of ATP on RNA priming from those on DNA synthesis, products synthesized processively by alpha-A2-polymerase were sized by gel filtration. Results indicate that essentially all products made on a gapped fd replicative form template in the presence of four dNTPs and 4 mM ATP result from the extension of preexisting DNA primers.(ABSTRACT TRUNCATED AT 250 WORDS)

. IntroductionNondividing cerebellar neurons have been shown to restore their chromosomal DNA structure much more slowly than intracerebral 9L brain tumour cells after irradiation with doses of 50-0 Gy (Wang and Wheeler 1978) . There are several possible explanations for this phenomenon . First, 50-0 Gy is certainly not a biologically relevant dose and at lower, more relevant doses the differences in the DNA repair kinetics between neurons and tumour cells might disappear . Secondly, permanently nondividing cells like neurons might require the induction of components of the repair system that are already available in the predominantly dividing tumour cells as part of their DNA replication machinery . Thirdly, there may be differences in the quantity, quality or types of repair enzymes found in dividing and nondividing cells . Lastly, the chromatin structure in nondividing cells may restrict the accessibility of these repair enzymes to the lesions or interfere with their operation at these lesions . Materials and methodsThe entire heads of anaesthetized, 240-280 g (3-4 month old), male Fisher 344 rats bearing 100-200 mg intracerebral 9L brain tumours were irradiated with single doses of 12-5 or 25 . 0 Gy of Cs-137 y-rays at a dose rate of 9-21 Gy/min . The remainder of the body was shielded with lead . At various times after irradiation, the rats were killed by decapitation and the tumour and cerebellum were removed . The tumour and cerebellum were dissociated into a single cell suspension at 4°C by mechanical means and the cell suspensions layered onto a lytic zone over a 10-30% alkaline sucrose gradient in two Beckman Ti-15 zonal rotors . After sedimentation the DNA was collected in 20 ml fractions, precipitated with perchloric acid, reacted with diaminobenzoic acid dihydrochloride and the amount of DNA in each fraction determined by spectrofluorometrically measuring the fluorescence emitted at 500 nm after excitation at 409 rim . Complete descriptions of the tumour implantation procedure, tumour growth characteristics, dissociation procedure, gradients, sedimentation conditions, typical sedimentation profiles and the molecular properties of the sedimenting DNA have been published (Wang and Wheeler 1978, Leith et al . 1975, Wheeler and Wallen 1980 .The percentage of the DNA with sedimentation coefficients >250S was calculated from the DNA sedimentation profiles of both irradiated and unirradiated

Rats bearing intracerebral 9L tumors were whole-brain irradiated with 1250 to 5000 rad, and the in situ DNA repair kinetics of the undifferentiated tumor cells and terminally differentiated cerebellar neurons were examined by alkaline sucrose gradient sedimentation in zonal rotors with gradient reorienting capability. Biphasic repair kinetics were observed for both tumor cells and cerebellar neurons. Quantitation and analysis of the slow phase of the repair process suggest that the dividing tumor cell genome is completely accessible to the enzymatic repair machinery, while it is possible that the genome of the permanently nondividing neuron may contain a region that is inaccessible to this repair machinery.